The cAMP-dependent protein kinase (PKA), the prime target of the second messenger cAMP in mammalian cells, is activated by the binding of cAMP to the regulatory subunit (R), of the molecule and results in the release of the active catalytic kinase subunit (C). There are several isoforms of both subunits of PKA. All members of the PKA family share significant sequence homology and perform signal transduction via protein phosphorylation. Almost all cell types express one or more isoforms of PKA. The protein inhibitor of PKA (PKI) acts by binding with high affinity to the substrate binding site of the free active catalytic subunit (Walsh D A (1990) Peptides and Protein Phosphorylation, CRC, Boca Raton Fla., pp. 43-84). In addition, PKI has been shown to export the C subunit from the nucleus (Fantozzi D A et al (1994) J Biol Chem 269: 2676-2686).
The endogenous inhibitor of cAMP-dependent protein kinase (PKA) is down-regulated in the kidneys from vitamin-D-replete chicks as compared to vitamin-D-deficient chicks. Screening of a vitamin-D-deficient chick kidney library resulted in the isolation of a 450-bp cDNA clone encoding the 76-amino acid protein kinase inhibitor (Marchetto G S and Henry H L (1995) Gene 158: 303-304). The deduced amino acid sequence of avian PKI shares 80% and 41% identity with the mammalian PKI alpha and PKI beta 1 isoforms, respectively.
Both avian PKI and human PKI alpha share conserved N-terminal sequences, including the pseudo-substrate site (18GRRNA22), which are required for potent inhibition of the catalytic subunit of PKA (Marchetto and Henry, supra). The amino acid phenylalanine at position 11 plays a critical role in PKI-C subunit binding (Baude et al (1994) J Biol Chem 269: 18128-18133).
Cell cycle synchrony studies suggests that PKI has an important role in the inhibition of nuclear C subunit activity that is required for cell cycle progression (Wen W et al (1995) J Biol Chem 270: 2041-2046). Microinjection of PKI alpha antibody prevented the cell cycle progression of serum-starved cells.
Disease and PKA
Human airway smooth muscle (ASM) contains beta 2-adrenergic receptors which, when stimulated, cause a rise in intracellular cAMP and activation of PKA, which in turn phosphorylates several cellular proteins, resulting in relaxation (Barnes P J (1993) Life Sci 52: 2101-2109). Activated PKA also acts in ASM cell proliferation, which contributes to increased airway resistance in bronchial asthma. Vasoactive intestinal peptide (VIP) selectively and potently inhibits human airway smooth muscle cell growth and multiplication, and nullifies the mitogenic effect of histamine, by a PKA-mediated mechanism. A deficiency of VIP may lead to ASM hyperplasia due to unopposed stimulation by endogenous mitogens. A selective inhibitor of PKA abolished the inhibitory effect of VIP (Maruno K et al (1995) Am J Physiol 268: 1047-1051).
Cystic fibrosis is caused by a mutation in the CFTR gene. CFTR, a chloride conducting membrane protein, is a substrate for PKA phosphorylation. This phosphorylation has been determined to be a step in the activation pathway (Drumm M L and Collins F S (1993) Mol Genet Med 3: 33-68).
Systemic lupus erythematosus (SLE) is an autoimmune disorder of indeterminate etiology characterized by a dysfunctional cellular immune response. T lymphocytes from subjects with active systemic lupus erythematosus exhibit reduced cAMP-inducible, PKA-dependent protein phosphorylation of several intracellular substrates compared with healthy and disease controls. Intact T cells from patients with severe SLE disease activity are impaired in PKA-catalyzed protein phosphorylation (Kammer G M et al (1994) J Clin Invest 94: 422-430). Variation in the concentration of PKA activity is an important element in modulating T cell proliferative responses. Induction of PKA activity in T cells inhibits anti-CD3 monoclonal antibody-induced T cell proliferation (Bauman G P et al (1994) Cell Immunol 158: 182-194).
The development of cross-resistance to many chemotherapeutic drugs, termed multidrug resistance (MDR), is one of the major reasons why cancer chemotherapy ultimately fails. MDR is often associated with over-expression of the MDR1 gene product, P-glycoprotein; a multifunctional drug transporter. MDR1 expression can be modulated by PKA, opening up the possibility of modulating MDR by selectively down-regulating the activity of PKA-dependent transcription factors which upregulate MDR1 expression. High levels of type I PKA occur in primary breast carcinomas and patients exhibiting this phenotype show decreased survival (Glazer R I and Rohlff C Breast Cancer Res Treat (1994) 31: 263-271).
The selective PKA inhibitors, 8-Cl-cAMP and Rp8-Cl-cAMPS! may be particularly useful for downregulating PKA-dependent MDR-associated transcription factors. These compounds downregulate transient expression of a reporter gene under the control of several MDR1 promoter elements (Glazer and RohIff, supra). 8-Cl-cAMP has recently been shown to decrease MDR1 expression in multidrug-resistant human breast cancer cells (Scala S et al (1995) J Clin Invest 96: 1026-1034).
PKA participates in the sequence of molecular events that underlie learning and memory. A peptide inhibitor of PKA disrupts associative learning when expressed in transgenic fruit flies (Drain P et al (1991) Neuron 6: 71-82). In mammalian systems, inhibitors of PKA were found to block hippocampal long-term potentiation, which is thought to be a mechanism for the establishment of explicit memory (Frey U et al (1993) Science 260: 1661-1664). In Alzheimer's disease, where memory loss is one of the earliest and most debilitating characteristics, tau proteins are phosphorylated by PKA. Tau degradation has been implicated in the pathogenesis of Alzheimer's disease and PKA phosphorylation of tau affects the rate of proteolytic degradation (Wang X et al (1996) Biochem Bioph Res Commun 219: 591-597).
PKA activity plays a role in many important biological systems. The selective inhibition of PKA may allow successful management of the diseases associated with PKA induced effects, such as cancer, memory disorders, and auto-immune diseases.